Abstract
1. To determine if structural domains are important for nicotinic acetylcholine receptor (nAChr) channel function, six mouse-Torpedo chimeric alpha-subunits were constructed (Fig. 2) and coexpressed with Torpedo californica beta-, gamma-, and delta-subunits in Xenopus laevis oocytes. 2. nAChRs containing a chimeric alpha-subunit were examined by voltage- and patch-clamp methods to determine their functional characteristics. Dose-response curves from voltage-clamped oocytes were used to estimate EC50's and Hill coefficients. Whole-cell currents were normalized against the alpha-bungarotoxin (alpha-BTX) binding sites to obtain normalized responses to acetylcholine (ACh). Open time constants at 4 microM ACh were used to examine single-channel behavior. 3. The EC50 for ACh was modulated by the N-terminal half of the alpha-subunit. When the Torpedo subunit sequence between position 1 and position 268 was replaced by mouse sequence, the EC50 shifted toward the value for the wild-type mouse subunit. Replacement of either the 1-159 or the 160-268 positions of the Torpedo sequence with the mouse sequence lowered the EC50. This suggests that at least two regions play a role in determining the EC50. 4. When the primary sequence (160-268) of the Torpedo alpha-subunit was introduced in the mouse alpha-subunit (T160-268), the expressed chimeric receptor was nonfunctional. The inverse chimera (M160-268) was functional and the open time constant and EC50 were similar to those of mouse but the normalized response was characteristic of Torpedo. 5. The normalized macroscopic response to ACh (300 microM) of the chimera containing the mouse alpha-subunit showed a ninefold increase relative to the Torpedo wild type. Receptors which contain the C terminal of the mouse alpha-subunit also show an increase in the maximum normalized current. Receptors with the alpha-subunit which contain the Torpedo C-terminal sequence have a lower normalized response. 6. The combined results suggest that AChR channel function is modulated by structural determinants within the primary sequence. These structural domains might modulate channel function through specific allosteric interactions. The lack of response of the T160-268 chimera suggests that a critical interaction essential for the coupling of agonist binding and channel gating was disrupted. This result suggests that the interaction of structural domains within the nAChR primary structure are essential for channel function and that these intractions could be very specific within different nAChR species.